Endomycorrhizal and rhizobial symbiosis: How much do they share?

Abstract

Legume plants enter two important endosymbioses – with soil fungi, forming phosphorus acquiring arbuscular mycorrhiza (AM), and with nitrogen-fixing bacteria, leading to the formation of nitrogen-fixing root nodules. Both symbioses have been studied extensively because these symbioses have great potential for agricultural applications. Although 80% of all living land plants form AM, the nitrogen-fixing root nodule symbiosis with rhizobia is almost exclusively restricted to legumes. Despite varying degree of differences in the morphological responses induced by both endosymbionts in the host plants, significant similarities in the development of both fungal and bacterial symbioses have been reported. The signal perception and signal transduction cascades that initiate nodulation and mycorrhization in legumes partially overlap. Legume genes have been identified that are required for the establishment of both AM and root nodule symbiosis and are referred to as the common SYM genes. Genetic dissection of the common SYM signal transduction pathway required for bacterial and fungal root endosymbiosis has not only unraveled the players involved but also provided a first glimpse at conservation and specialization of signaling cascades essential for nodulation and mycorrhiza development. Based on the observation of common signaling cascades, it is tempting to speculate that the root nodule symbiosis, where fossil records date back to the late Cretaceaous, adopted and subsequently modified more ancient signal transduction pathways leading to AM formation, having already been in place 400 million years ago. This review discusses the common aspects of recognition of mycorrhizal fungi and Rhizobium by the host, and further signal transduction that leads to an effective symbiosis.     

The Medicago truncatula lysin [corrected] motif-receptor-like kinase gene family includes NFP and new nodule-expressed genes

Rhizobial Nod factors are key symbiotic signals responsible for starting the nodulation process in host legume plants. Of the six Medicago truncatula genes controlling a Nod factor signaling pathway, Nod Factor Perception (NFP) was reported as a candidate Nod factor receptor gene. Here, we provide further evidence for this by showing that NFP is a lysin [corrected] motif (LysM)-receptor-like kinase (RLK). NFP was shown both to be expressed in association with infection thread development and to be involved in the infection process. Consistent with deviations from conserved kinase domain sequences, NFP did not show autophosphorylation activity, suggesting that NFP needs to associate with an active kinase or has unusual functional characteristics different from classical kinases. Identification of nine new M. truncatula LysM-RLK genes revealed a larger family than in the nonlegumes Arabidopsis (Arabidopsis thaliana) or rice (Oryza sativa) of at least 17 members that can be divided into three subfamilies. Three LysM domains could be structurally predicted for all M. truncatula LysM-RLK proteins, whereas one subfamily, which includes NFP, was characterized by deviations from conserved kinase sequences. Most of the newly identified genes were found to be expressed in roots and nodules, suggesting this class of receptors may be more extensively involved in nodulation than was previously known.     

Molecular evolution of lysin motif-type receptor-like kinases in plants

The lysin motif (LysM) domain is an ancient and ubiquitous protein module that binds peptidoglycan and structurally related molecules. A genomic survey in a large number of species spanning all kingdoms reveals that the combination of LysM and receptor kinase domains is present exclusively in plants. However, the particular biological functions and molecular evolution of this gene family remain largely unknown. We show that LysM domains in plant LysM proteins are highly diversified and that a minimum of six distinct types of LysM motifs exist in plant LysM kinase proteins and five additional types of LysM motifs exist in nonkinase plant LysM proteins. Further, motif similarities suggest that plant LysM motifs are ancient. Although phylogenetic signals are not sufficient to resolve the earliest relationships, plant LysM motifs may have arisen through common ancestry with LysM motifs in other kingdoms. Within plants, the gene family has evolved through local and segmental duplications. The family has undergone further duplication and diversification in legumes, where some LysM kinase genes function as receptors for bacterial nodulation factor. Two pairs of homeologous regions were identified in soybean (Glycine max) based on microsynteny and fluorescence in situ hybridization. Expression data show that most plant LysM kinase genes are expressed predominantly in the root and that orthologous LysM kinase genes share similar tissue expression patterns. We also examined synteny around plant LysM kinase genes to help reconstruct scenarios for the evolution of this important gene family.     

Long-distance transport of signals during symbiosis: Are nodule formation and mycorrhization autoregulated in a similar way?

Legumes enter nodule symbioses with nitrogen-fixing bacteria (rhizobia), whereas most flowering plants establish symbiotic associations with arbuscular mycorrhizal (AM) fungi. Once first steps of symbiosis are initiated, nodule formation and mycorrhization in legumes is negatively controlled by a shoot-derived inhibitor (SDI), a phenomenon termed autoregulation. According to current views, autoregulation of nodulation and mycorrhization in legumes is regulated in a similar way. CLE peptides induced in response to rhizobial nodulation signals (Nod factors) have been proposed to represent the ascending long-distance signals to the shoot. Although not proven yet, these CLE peptides are likely perceived by leucine-rich repeat (LRR) autoregulation receptor kinases in the shoot. Autoregulation of mycorrhization in non-legumes is reminiscent to the phenomenon of “systemic acquired resistance” in plant-pathogen interactions.Key words: arbuscular mycorrhiza, autoregulation, CLE peptides, mutant, nodulation, split-root systemUnder natural conditions, growth of plants is often limited by the availability of nutrients such as nitrogen and phosphorous. Plants have therefore developed strategies to acquire nutrients with the help of soil microorganisms. Most land plants enter mutualistic root symbioses with arbuscular mycorrhizal (AM) fungi, whereas legumes form special root nodules containing nitrogen-fixing bacteria, so-called rhizobia.^1–4 Establishment and maintenance of symbiosis requires plant resources, such as photosynthetically assimilated carbon. To minimize these costs, host plants are able to control the degree of their symbiotic interactions. Above a critical threshold level further establishment of symbiosis is restricted—a feedback phenomenon termed autoregulation of symbiosis. Autoregulation can be experimentally demonstrated in split-root systems. When legume roots are already infected by rhizobia on one side of a split-root, further nodule development is “systemically” inhibited on the other side. Similarly, prior colonization of split-roots by AM fungi on one half suppresses later fungal root colonization on the other half. Hence, important elements of the symbiotic autoregulation circuit are not only localized in roots, but also in aerial parts of the plant, implicating transport of signals in vascular bundles (Fig. 1). Whereas autoregulation of nodulation in legumes has been studied for many decades,^5–9 the first publications clearly stating a shoot-controlled autoregulation of mycorrhization in split-root systems appeared in 2000 for the non-legume barley (Hordeum vulgare) and thereafter for alfalfa (Medicago sativa) and soybean (Glycine max).^10–13 The data from these split-root experiments are supported by the findings that supernodulating (or hypernodulating) loss-of-autoregulation mutants displayed either an increased degree of AM colonization and/or a higher abundance of arbuscules.^14–16Open in a separate windowFigure 1Proposed model of shoot-controlled autoregulation of symbiosis in a split-root system. Prior infection of root A by rhizobia or AM fungi systemically suppresses later establishment of symbiosis in root B. Expression of specific CLE peptides (and/or other peptide hormones) is induced in response to rhizobial nodulation signals (Nod factors) and perhaps also in response to colonization by AM fungi (stage 1). The CLE peptides (and/or other signals) are then presumed to be transported in the xylem to the shoot, where they are perceived by leucine-rich repeat (LRR) autoregulation receptor kinases (stage 2). As a result of autoregulation signaling in the shoot, an unknown shoot-derived inhibitor (SDI) is produced (stage 3) and transported as a phloem-mobile signal to the root. Perception and action of the SDI signal in roots would then inhibit nodulation and root colonization by AM fungi (stage 4).     

A dominant function of CCaMK in intracellular accommodation of bacterial and fungal endosymbionts

In legumes, Ca²⁺/calmodulin‐dependent protein kinase (CCaMK) is a component of the common symbiosis genes that are required for both root nodule (RN) and arbuscular mycorrhiza (AM) symbioses and is thought to be a decoder of Ca²⁺ spiking, one of the earliest cellular responses to microbial signals. A gain‐of‐function mutation of CCaMK has been shown to induce spontaneous nodulation without rhizobia, but the significance of CCaMK activation in bacterial and/or fungal infection processes is not fully understood. Here we show that a gain‐of‐function CCaMK^T265D suppresses loss‐of‐function mutations of common symbiosis genes required for the generation of Ca²⁺ spiking, not only for nodule organogenesis but also for successful infection of rhizobia and AM fungi, demonstrating that the common symbiosis genes upstream of Ca²⁺ spiking are required solely to activate CCaMK. In RN symbiosis, however, CCaMK^T265D induced nodule organogenesis, but not rhizobial infection, on Nod factor receptor (NFRs) mutants. We propose a model of symbiotic signaling in host legume plants, in which CCaMK plays a key role in the coordinated induction of infection thread formation and nodule organogenesis.     

丛枝菌根真菌脂类代谢对共生信号调控的响应和反馈

丛枝菌根(AM)真菌是自然生态系统中分布最为广泛的真菌之一,在自然界物质循环和能量流动中发挥着重要作用。经过长期的协同进化,AM真菌和宿主植物之间形成了完美的互惠互利的共生关系,而真菌的脂类代谢可能是揭示共生秘密的关键所在。本文综述了AM真菌脂类代谢在共生关系建立和维持中关键作用的最新研究进展,重点探讨了AM真菌脂类代谢对共生信号调控的响应和反馈机制,主要包括:AM真菌脂类存储和释放对共生和非共生状态的响应,以及脂类代谢产物变化与共生营养传递之间的关系;脂类分解过程在共生建立初期对信号分子调控发生的响应,以及相应的物质转化和能量代谢;菌根共生互惠互利关系维持中,真菌脂类代谢与信号分子交流通道的相互渗透和影响。本文对于理解菌根共生机制,促进菌根在生产中的应用具有促进作用。     

Successful joint ventures of plants: arbuscular mycorrhiza and beyond

Among the oldest symbiotic associations of plants are arbuscular mycorrhiza (AM) with fungi of the phylum Glomeromycota. Although many of the symbiotic signaling components have been identified on the side of the plant, AM fungi have long evaded genetic analysis owing to their strict biotrophy and their exceptional genetics. Recently, the identification of the fungal symbiosis signal (Myc factor) and of a corresponding Myc factor receptor, and new insights into AM fungal genetics, have opened new avenues to address early communication and functional aspects of AM symbiosis. These advances will pave the way for breeding programs towards adapted AM fungi for crop production, and will shed light on the ecology and evolution of this remarkably successful symbiosis.     

The Jasmonic Acid Signalling Pathway Restricts the Development of the Arbuscular Mycorrhizal Association in Tomato

The role of the jasmonate signalling pathway in modulating the establishment of the arbuscular mycorrhiza (AM) symbiosis between tomato plants and Glomus intraradices fungus was studied. The consequences of AM formation due to the blockage of the jasmonate signalling pathway were studied in experiments with plant mutants impaired in JA perception. The tomato jai-1 mutant (jasmonic acid insensitive 1) failed to regulate colonization and was more susceptible to fungal infection, showing accelerated colonization. The frequency and the intensity of fungal colonization were greatly increased in the jai-1 insensitive mutant plants. In parallel, the systemic effects on mycorrhization due to the activation of the jasmonate signalling pathway by foliar application of MeJA were evaluated and histochemical and molecular parameters of mycorrhizal intensity and efficiency were measured. Histochemical determination of fungal infectivity and fungal alkaline phosphatase activity reveal that the systemic application of MeJA was effective in reducing mycorrhization and mainly affected fungal phosphate metabolism and arbuscule formation, analyzed by the expression of GiALP and the AM-specific gene LePT4, respectively. The results of the present study clearly show that JA participates in the susceptibility of tomato to infection by arbuscular mycorrhizal fungi, and it seems that arbuscular colonization in tomato is tightly controlled by the jasmonate signalling pathway.     

植物激素调控丛枝菌根发育的作用机制研究进展

丛枝菌根（arbuscular mycorrhiza，AM）是土壤中AM真菌和绝大多数维管植物根系长期进化过程中相互识别、相互作用形成的互利共生体。AM的发育与功能效应依赖AM真菌-寄主植物之间精准的“分子对话”，同时受到环境条件特别是土壤养分水平、干旱和盐渍化的制约。植物激素作为低浓度的小分子有机物，是参与调控AM共生过程的重要信号分子。其中，主要有9种植物激素参与AM发育过程且分工各有不同：独脚金内酯（strigolactones，SLs）参与AM真菌-寄主植物之间最初的共生识别，脱落酸（abscisic acid，ABA）和油菜素内酯（brassinosteroid，BR）促进前期的菌丝入侵，但水杨酸（salicylic acid，SA）和乙烯（ethylene，ET）抑制前期的菌丝入侵，生长素（auxin，Aux）、ABA和BR促进随后的丛枝形成而ET和赤霉素（gibberellin，GA）的作用则相反，茉莉酸（jasmonic acid，JA）对菌丝入侵与丛枝形成均可能存在正调控或负调控作用。目前细胞分裂素（cytokinin，CTK）在AM发育中的作用尚不明确。更为复杂的是，通常植物激素信号之间的交叉互作决定AM的发育进程。本文针对AM发育过程总结了不同植物激素的调控作用特点和不同植物激素信号之间的互作（协同或拮抗），以及胁迫条件下不同植物激素信号的可能调控机制。深入研究和系统阐明植物激素调控AM真菌-寄主植物共生的生理/分子机制，将有助于促进生物共生学理论研究及菌根技术的应用。     

Four genes of Medicago truncatula controlling components of a nod factor transduction pathway

Rhizobium nodulation (Nod) factors are lipo-chitooligosaccharides that act as symbiotic signals, eliciting several key developmental responses in the roots of legume hosts. Using nodulation-defective mutants of Medicago truncatula, we have started to dissect the genetic control of Nod factor transduction. Mutants in four genes (DMI1, DMI2, DMI3, and NSP) were pleiotropically affected in Nod factor responses, indicating that these genes are required for a Nod factor-activated signal transduction pathway that leads to symbiotic responses such as root hair deformations, expressions of nodulin genes, and cortical cell divisions. Mutant analysis also provides evidence that Nod factors have a dual effect on the growth of root hair: inhibition of endogenous (plant) tip growth, and elicitation of a novel tip growth dependent on (bacterial) Nod factors. dmi1, dmi2, and dmi3 mutants are also unable to establish a symbiotic association with endomycorrhizal fungi, indicating that there are at least three common steps to nodulation and endomycorrhization in M. truncatula and providing further evidence for a common signaling pathway between nodulation and mycorrhization.     

High effectiveness of exotic arbuscular mycorrhizal fungi is reflected in improved rhizobial symbiosis and trehalose turnover in Cajanus cajan genotypes grown under salinity stress

Arbuscular mycorrhizal fungi (AMF) improve functioning of legume-Rhizobium symbiosis under salinity. However, plant responses to mycorrhization vary depending on the plant and fungal species. The current study aimed to compare the effectiveness of a native inoculum from saline soil and two exotic isolates, Funneliformis mosseae and Rhizophagus irregularis on two Cajanus cajan (pigeonpea) genotypes (Paras, Pusa 2002) subjected to NaCl stress. Salinity depleted nodulation and nutrient status in both genotypes with higher negative effects in Paras. Although all AM fungi improved growth, R. irregularis performed better by promoting higher biomass accumulation, nodulation, N₂ fixation and N, P uptake which correlated with higher AM colonization. R. irregularis inoculated plants also accumulated higher trehalose in nodules due to decreased trehalase and increased trehalose-6-P synthase, trehalose-6-phosphatase activities. The results suggest that higher stability of R. irregularis-pigeonpea symbiosis under salt stress makes it an effective ameliorator for overcoming salt stress in pigeonpea.     

A short LysM protein with high molecular diversity from an arbuscular mycorrhizal fungus,Rhizophagus irregularis

Arbuscular mycorrhizal (AM) fungi form an intimate symbiosis with roots of more than 80% of land plants without eliciting a significant defense response, and how they do so is yet to be determined. Typically, plants mount a defense response upon sensing chitin in fungal walls, and to counteract this response, plant-pathogenic fungi secrete small effector proteins with chitin-binding LysM domains. In the AM fungus, Rhizophagus irregularis, a small, putatively-secreted LysM protein, which we refer to as RiSLM, is among the most highly expressed effector-like proteins during symbiosis. Here, we show that RiSLM expression is reduced during non-functional symbiosis with Medicago mutants, mtpt4-2 and vapyrin. We demonstrate that RiSLM can bind to both chitin and chitosan, and we model the protein-ligand interaction to identify possible binding sites. Finally, we have identified RiSLM homologs in five published R. irregularis isolate genomes and demonstrate that the gene is subject to a high rate of evolution and is experiencing positive selection, while still conserving putative function. Our results present important clues for elucidating a role for a LysM effector, RiSLM, in AM symbiosis.     

Interaction of Medicago truncatula Lysin Motif Receptor-Like Kinases,NFP and LYK3, Produced in Nicotiana benthamiana Induces Defence-Like Responses

Receptor(-like) kinases with Lysin Motif (LysM) domains in their extracellular region play crucial roles during plant interactions with microorganisms; e.g. Arabidopsis thaliana CERK1 activates innate immunity upon perception of fungal chitin/chitooligosaccharides, whereas Medicago truncatula NFP and LYK3 mediate signalling upon perception of bacterial lipo-chitooligosaccharides, termed Nod factors, during the establishment of mutualism with nitrogen-fixing rhizobia. However, little is still known about the exact activation and signalling mechanisms of MtNFP and MtLYK3. We aimed at investigating putative molecular interactions of MtNFP and MtLYK3 produced in Nicotiana benthamiana. Surprisingly, heterologous co-production of these proteins resulted in an induction of defence-like responses, which included defence-related gene expression, accumulation of phenolic compounds, and cell death. Similar defence-like responses were observed upon production of AtCERK1 in N. benthamiana leaves. Production of either MtNFP or MtLYK3 alone or their co-production with other unrelated receptor(-like) kinases did not induce cell death in N. benthamiana, indicating that a functional interaction between these LysM receptor-like kinases is required for triggering this response. Importantly, structure-function studies revealed that the MtNFP intracellular region, specific features of the MtLYK3 intracellular region (including several putative phosphorylation sites), and MtLYK3 and AtCERK1 kinase activity were indispensable for cell death induction, thereby mimicking the structural requirements of nodulation or chitin-induced signalling. The observed similarity of N. benthamiana response to MtNFP and MtLYK3 co-production and AtCERK1 production suggests the existence of parallels between Nod factor-induced and chitin-induced signalling mediated by the respective LysM receptor(-like) kinases. Notably, the conserved structural requirements for MtNFP and MtLYK3 biological activity in M. truncatula (nodulation) and in N. benthamiana (cell death induction) indicates the relevance of the latter system for studies on these, and potentially other symbiotic LysM receptor-like kinases.     

Regulation of arbuscular mycorrhization by carbon. The symbiotic interaction cannot be improved by increased carbon availability accomplished by root-specifically enhanced invertase activity

The mutualistic interaction in arbuscular mycorrhiza (AM) is characterized by an exchange of mineral nutrients and carbon. The major benefit of AM, which is the supply of phosphate to the plant, and the stimulation of mycorrhization by low phosphate fertilization has been well studied. However, less is known about the regulatory function of carbon availability on AM formation. Here the effect of enhanced levels of hexoses in the root, the main form of carbohydrate used by the fungus, on AM formation was analyzed. Modulation of the root carbohydrate status was performed by expressing genes encoding a yeast (Saccharomyces cerevisiae)-derived invertase, which was directed to different subcellular locations. Using tobacco (Nicotiana tabacum) alcc::wINV plants, the yeast invertase was induced in the whole root system or in root parts. Despite increased hexose levels in these roots, we did not detect any effect on the colonization with Glomus intraradices analyzed by assessment of fungal structures and the level of fungus-specific palmitvaccenic acid, indicative for the fungal carbon supply, or the plant phosphate content. Roots of Medicago truncatula, transformed to express genes encoding an apoplast-, cytosol-, or vacuolar-located yeast-derived invertase, had increased hexose-to-sucrose ratios compared to beta-glucuronidase-transformed roots. However, transformations with the invertase genes did not affect mycorrhization. These data suggest the carbohydrate supply in AM cannot be improved by root-specifically increased hexose levels, implying that under normal conditions sufficient carbon is available in mycorrhizal roots. In contrast, tobacco rolC::ppa plants with defective phloem loading and tobacco pyk10::InvInh plants with decreased acid invertase activity in roots exhibited a diminished mycorrhization.     

A novel nuclear protein interacts with the symbiotic DMI3 calcium- and calmodulin-dependent protein kinase of Medicago truncatula

Many higher plants establish symbiotic relationships with arbuscular mycorrhizal (AM) fungi that improve their ability to acquire nutrients from the soil. In addition to establishing AM symbiosis, legumes also enter into a nitrogen-fixing symbiosis with bacteria known as rhizobia that results in the formation of root nodules. Several genes involved in the perception and transduction of bacterial symbiotic signals named "Nod factors" have been cloned recently in model legumes through forward genetic approaches. Among them, DMI3 (Doesn't Make Infections 3) is a calcium- and calmodulin-dependent kinase required for the establishment of both nodulation and AM symbiosis. We have identified, by a yeast two-hybrid system, a novel protein interacting with DMI3 named IPD3 (Interacting Protein of DMI3). IPD3 is predicted to interact with DMI3 through a C-terminal coiled-coil domain. Chimeric IPD3::GFP is localized to the nucleus of transformed Medicago truncatula root cells, in which split yellow fluorescent protein assays suggest that IPD3 and DMI3 physically interact in Nicotiana benthamiana. Like DMI3, IPD3 is extremely well conserved among the angiosperms and is absent from Arabidopsis. Despite this high level of conservation, none of the homologous proteins have a demonstrated biological or biochemical function. This work provides the first evidence of the involvement of IPD3 in a nuclear interaction with DMI3.     

The LysM receptor kinase CERK1 mediates bacterial perception in Arabidopsis

Plants use pattern recognition receptors (PRRs) to perceive pathogen-associated molecular pattern (PAMPs) and initiate defence responses. PAMP-triggered immunity (PTI) plays an important role in general resistance, and constrains the growth of most microbes on plants. Despite the importance of PRRs in plant immunity, the vast majority of them remain to be identified. We recently showed that the Arabidopsis LysM receptor kinase CERK1 is required not only for chitin signalling and fungal resistance, but plays an essential role in restricting bacterial growth on plants. We proposed that CERK1 may mediate the perception of a bacterial PAMP, or an endogenous plant cell wall component released during infection, through its extracellular carbohydrate-binding LysM-motifs. Here we report reduced activation of a PAMP-induced defence response on plants lacking the CERK1 gene after treatment with crude bacterial extracts. This demonstrates that CERK1 mediates perception of an unknown bacterial PAMP in Arabidopsis.Key words: PAMP, PRR, PTI, LysM, chitin, bacteria, carbohydrate